Hantaviruses are found throughout most of the Americas and are the causative agents of Hantavirus Pulmonary Syndrome (HPS). HPS has a case fatality rate of 40 percent and there is currently no cure for this disease or licensed vaccines for the prevention of hantavirus infections. The lack of an effective treatment for hantavirus infection is partially attributable to a poor understanding of how these viruses gain entry to their host cell. Thus, the aim of this research application is to dissect the molecular entry pathway of a representative Hantavirus, Andes virus (ANDV), with the broad goal of identifying potential therapeutic targets. This two year application is designed to carry forward a current human haploid screening project that has already identified several clonal cell lines differentially refractory to a replication competent recombinant vesicular stomatitis virus bearing only the ANDV envelope (rVSV:ANDV). These cell lines were cloned from an insertionally-mutagenized population of human haploid cells following a lethal selection with rVSV:ANDV and tested positive for wild type rVSV susceptibility. This indicated that the defects in infectivity wre determined by the ANDV protein envelope and likely at the point of entry. Aim 1 of this application is to identify and verify new human factors required for the entry of Andes Virus. This aim compiles the use of standard molecular biology techniques and cutting edge 454 deep sequencing technologies to map the location of genes required for Andes Virus entry. By ranking the number of independent insertions into known genes within these two populations, it has been possible to statistically identify genes important for viral entry. The importance of these genes will need to be validated, and the final portion of Aim 1 describes a strategy for creating expression knockdowns and knockouts of these genes to retest infectivity with wild type BSL-3 Andes Virus. Aim 2 will establish the point of defect along the Andes Virus entry pathway (binding, endocytosis, fusion), and test if other bunyaviruses utilize any of the identified host factors during their entry. Together, these aims serve to complete a body of research that will yield basic scientific data that may translate to clinical applications for the treatment of HPS an prevention of ANDV infection.